Project Summary/ Abstract: An acute injury, such as myocardial infarction (MI), induces exaggerated production of myeloid cells in the bone marrow. These newly generated myeloid cells are inflammatory and are crucial in disease pathogenesis, exacerbating underlying processes such as atherosclerosis. However, the systemic long distance signals and molecular mechanisms that trigger myelopoiesis in the bone marrow after an ischemic event at a distant organ, such as the heart, are poorly understood. Our preliminary data revealed that, following an MI, bone marrow hematopoietic stem and progenitor cells (HSPC) relocated into the vascular niches. Vascular niches confer distinct microenvironments promoting HSPC proliferation and differentiation. In investigating the long-distance signals produced after MI, we found increased levels of platelet-derived extracellular vesicles (pEV) in the blood of patients and mice after MI. These pEV increased the expression of PU.1, which is a myeloid transcription factor, and the receptor for interleukin-3 (IL-3R), which increases myeloid cell generation, in bone marrow HSPC. Additionally, pEV isolated from mice with MI contained high levels of miR-499, which increased bone marrow HSPC proliferation and differentiation in vitro. From these observations, we hypothesize that increased miR-499 in pEV following MI relocates HSPC to active vascular niches in the bone marrow, where they proliferate and differentiate into myeloid cells. We will test this hypothesis in two specific aims: (1) We will determine the role of pEV in relocation of HSPC to the vascular niches in the bone marrow. (2) We will investigate if increased miR-499 expression in pEV augments myelopoiesis and atherosclerosis after MI. To test the hypothesis, we will photoconvert KikGR+ HSPC, use mice deficient of miR-499 and two different mouse models of atherosclerosis. We will characterize pEV in patients and mice with MI using flow cytometry, ImageStream and NanoSight technologies. Additionally, we will determine the proliferation of HSPC exposed to pEV isolated from miR- 499+/+ and miR-499-/- mice after MI in vitro and in vivo. The proposed grant application will further our understanding of the mechanisms of MI-induced myelopoiesis, and explore new therapeutic avenues to diminish inflammation and atherosclerosis after MI.